The hedgehog (hh) family members control many aspects of development in both vertebrates and invertebrates. Hh signaling is associated with important biological phenomena such as patterning, cell growth, and morphogenesis. Abnormal activation of this pathway has been observed in several types of human cancers. The seven-pass transmembrane protein Smoothened (Smo) is required in both insects and mammals for transduction of the Hh signal. Our strategy is to use Drosophila as a simple and genetically tractable model system to explore the mechanisms of Hh signal transduction. The long-term goal of our research is to elucidate how Hh signals are sensed and transmitted to control downstream biological events that ultimately govern cell growth and patterning. We showed that Smo transduces the Hh signals by directly recruiting a Costal2-Fused (Cos2-Fu) complex. We also showed that Smo activation requires phosphorylation by protein kinase A (PKA) and casein kinase I (CKI), and that phosphorylation leads to increased Smo cell-surface levels and signaling activity. We recently uncovered a feedback mechanism by which Fu promotes Smo hyperphosphorylation and cell-surface accumulation by antagonizing Cos2. These findings provide new tools and hypotheses to investigate the mechanisms of Hh signaling at the cell membrane. In this project, our central hypothesis is that Smo cell-surface accumulation and signaling activity are regulated by multiple kinases, while phosphatase(s) act as inhibitory components to attenuate Smo activation. To test our hypothesis, we will use a combination of genetic and biochemical approaches in three Specific Aims: 1) To further determine if differential Smo phosphorylation transduces gradient Hh activity;2) to investigate the molecular mechanisms by which G protein-coupled receptor kinase 2 (GRK2) is involved in Hh signaling;and 3) to determine the role of phosphatase in Smo dephosphorylation and inactivation. HEALTH RELEVANCE Abnormal Smoothened (Smo) activation results cancers such as basal cell carcinoma (BCC) and medulloblastoma. Investigation of the Smo signaling mechanisms will provide both insights into fundamental developmental problems and new avenues for developing diagnostic tools and therapeutical treatments of cancers. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Project Narrative